(552) Chemoattraction of Pseudomonas aeruginosa PAO1 to Bovine Bile in a Novel, Cystic Fibrosis-Derived Bronchial Epithelial Cell Model

Background: Persistent and ultimately deteriorating multi-year bacterial pulmonary infections are the major determinant of lifespan in people with cystic fibrosis (pwCF), accounting for over 90% of the morbidity and mortality associated with the disease. Those infections are dominated in adulthood by the opportunistic pathogen, Pseudomonas aeruginosa (Pa). As with other advancing respiratory illnesses, pwCF also frequently suffer from gastroesophageal reflux disease (GERD), including bile aspiration into the lung. GERD is a major co-morbidity factor in pwCF, with a reported prevalence of 35–81% in affected individuals. Indeed, despite the inherent bactericidal nature of bile its association with the early airway tract acquisition of Pa in pwCF is well known. Moreover, in vitro studies show that bile causes Pa to adapt a chronic lifestyle stimulates Pa virulence, facilitates colonization, and further enhances infection through stimulating biofilm formation, Type VI secretion, efflux pump expression, quorum sensing, and antibiotic tolerance. We hypothesized that Pa is chemoattracted to bile in the lung environment. To better mimic the in situ bacterial chemoattractions during pathogenesis, we have established a model that builds on the principles of Adler’s classic assay system.

Methods: We developed a novel chemotaxis experimental system mimicking the lung environment utilizing CF-derived bronchial epithelial (CFBE) cells which allowed for the evaluation of Pa (strain PAO1) chemotaxis in a physiological scenario superior to the standard in vitro systems. We performed qualitative and quantitative chemotaxis tests using this new experimental system, as well as traditional Adler’s microcapillary assays to investigate the chemoattraction response of Pa toward physiologically-relevant concentrations of bovine bile.

Results: Our studies demonstrated that bovine bile is a chemoattractant for P. aeruginosa PAO1 and is positively correlated with bile concentration. Moreover, using our CFBE-based model evaluating the chemotaxis response of Green-fluorescence protein (GFP) labeled P. aeruginosa PAO1 revealed its attraction toward bile in a concentration- and time-dependent manner.

Conclusions: Our experimental model demonstrates how Pa colonization in the CF lung may be preceded or enhanced by chemotactic responses to bile. These results further buttress the hypothesis that bile likely contributes to the colonization and pathogenesis of Pa in the lung, particularly in pwCF. This experimental system can facilitate studying Pa, and other motile CF opportunistic bacterial pathogens (e.g., Burkholderia cepacia complex and Stenotrophomonas maltophilia), chemotaxis, initial binding, as well as colonization at the same time. In addition, this CF-derived model could be useful for studies with eukaryotic cells, such as neutrophils, in the context of CF lung. This is a promising start and additional adjustments to this novel approach can be developed to closely represent the CF lung niche. Subsequent studies to assess the Pa chemoreceptor(s) involved in the PAO1 response, particularly toward individual constituents of bile. as well as an assessment of whether other Pa strains also exhibit chemoattraction, might cast additional light on Pa pathogenesis in the CF lung.

Acknowledgements: We are grateful to Valentina Sedlacek and Rachel Dokko for their technical assistance. This study was supported by the US Cystic Fibrosis Foundation, HILL17P0 for J.E.H., and the NIH and CF Foundation, STANTO19R0. R01 HL151385-01A for B.A.S.

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